GSM6657988: RAD21* site2 0h; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
RAD21
Cell type
Cell type Class
Digestive tract
Cell type
DLD-1
Primary Tissue
Colon
Tissue Diagnosis
Adenocarcinoma
Attributes by original data submitter
Sample
source_name
DLD-1
cell line
DLD-1
cell type
adenocarcinoma, colon epithelium
time point
0h
antibody
mAbs against IEEPSR C-terminus
Sequenced DNA Library
library_name
GSM6657988
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For ChIP-On-ChEPseq, designed to extract pure fixed chromatin under stringent chaotropic conditions (PMID: 25101823, PMID: 36179046), 10 to 20 million cells were resuspended in 1 ml of nuclei lysis buffer (25 mM Tris-HCl, 85 mM KCl, 0.1% Triton-X100, pH 7.4, Roche PIC), centrifuged at 2300 g, resuspended again in the same buffer containing 200 µg/ml RNase A, and the samples were incubated in thermomixer at 37˚C for 15 min at 1000 rpm. After that, samples were spun at 2300 g for 5 min at 4˚C, pellets were resuspended in 500 µl of nuclei disrupting buffer (4% SDS, 50 mM Tris-HCl, 10 mM EDTA, pH 7.4), and mixed with 1.5 ml of urea buffer (8 M urea, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4). After gentle mixing leading to the clarification of the solution, it was centrifuged at 16,100 g for 30 min at 23°C. After decanting of the supernatant, resulting pure chromatin was washed twice with sonication buffer (20 mM Tris-HCl, 150 mM NaCl, 1% Triton-X100, 0.1% Na deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM PMSF) , and samples were reconstituted with sonication buffer to 1 ml volume. Chromatin fragmentation was done in 2-ml tubes in Covaris S220 with the following settings: 100W peak incident power, 10% duty factor, 10W average incident power, 30/30s duration/delay, 200 cycles/burst, 30 repeats. TruSeq Nano DNA library kit (Illumina).